The invention concerns a recombinant DNA including a nucleotide sequence coding for a specific polypeptide under the control of an adenovirus promoter, the vectors containing this recombinant DNA, the eucaryotic cells transformed by this recombinant DNA, the excretion products of these transformed cells and their applications, notably to the constitution of vaccines.
Human adenoviruses possess a long (around 36,000 bp) linear and double-stranded genome which codes for at least 30 proteins. The viral cycle, in the course of the infection of permissive cells, is divided into two phases, early and late. It is known that the four regions of the viral genome expressed in the early phase are called regions E1, E2, E3, and E4, whose respective positions in the whole genome are schematically represented in FIG. 1. The E1 region, situated at the left end of the genome, is itself divided into two regions, E1A and E1B. The passage from the early phase to the late phase, marked by the replication of the viral DNA, is characterized by an abrupt change in the genetic program of the virus. The expression of certain early genes is repressed while the transcription of the late genes is accomplished principally via only one promoter, the major late promoter (FIG. 1). In addition, a strong repression of protein synthesis of the host cell may be observed (1).
The genetic organization of type 2 or 5 human adenovirus (Ad2, Ad5) is sufficiently well known that their genome may be manipulated in vitro and its use as a vector for the expression of a foreign gene in an animal cell in culture has already been envisaged. Indeed it is known that the E3 region, which represents 6% of the genome, is not essential in vitro and may therefore be substituted in its entirety (2). The size of the foreign DNA fragment which it is possible to insert into the genome of these viruses, is large. In fact, the virus may encapsidate a genome whose length exceeds by 5% that of the wild genome.
Different vectors derived from adenoviruses of type 2 or 5 have therefore been constructed. In these recombinants, the foreign gene was expressed under the control of the major late promoter. This has permitted the obtaining in certain cases of a synthesis of the protein coded by a foreign gene at a level comparable to that of the late viral proteins (4, 5, 6, 7). This being the case, it results from the preceding that the expression of the foreign gene under the control of the late promoter can only manifest itself in the late phase of the viral cycle.
The invention results from the observation that the promoter of the early region E1A of the genome of an adenovirus (hereafter designated simply as xe2x80x9cE1A promoterxe2x80x9d) could control in a particularly effective manner the expression of a heterologous gene (that is, foreign vis-a-vis the genes normally associated with it in the adenovirus) or more generally of a heterologous nucleotide sequence coding for a polypeptide sequence whose expression is sought. In other words, the E1A promoter acts like a strong promoter, and this more particularly when the E1A promoter combined with the heterologous coding sequence is inserted into a viral vector.
The invention therefore concerns in a general fashion a recombinant DNA for the transformation of eucaryotic cell lines, notably human or animal, chosen from among which are infectable by the adenoviruses or whose endogenous polymerases are likely to recognize the adenovirus promoters, this recombinant DNA being, in addition, modified by an insertion nucleic acid containing a nucleotide sequence coding for a polypeptide sequence whose expression in the said cell lines is sought. This recombinant DNA is more particularly characterized by the fact that the said insertion sequence is placed under the direct control of the early promoter of the E1A region of the genome of the adenovirus.
Preferably, this recombinant DNA is incorporated into a replicatable vector in the said cell lines or associated by genetic recombination with such a vector.
Being a viral vector, notably one derived from adenovirus, equally offers the advantages attached to the E1A region of the adenoviruses, namely that its expression is constitutive and permanent all during the viral cycle (8, 9).
A particularly prefered form of the recombinant DNA according to the invention is characterized by the fact that it includes, xe2x80x98downstreamxe2x80x99 of the insertion nucleic acid, in the direction of transcription, a defective adenovirus genome including nevertheless all of those of the essential sequences necessary to the replication of the corresponding adenovirus, which are normally situated xe2x80x98downstreamxe2x80x99 of the genes normally under the direct control of the E1A early promoter in the said genome.
Advantageously, the defective adenovirus genome with which the recombinant DNA conforming to the invention is associated, is constituted of complete adenovirus genome, deprived however of the anterior part of the E1A region of the viral genome, notably of its 0-2.6% fragment (the percentage expressed relative to the total size of the adenovirus genome).
The recombinant DNAs of the invention, associated with the elements of vectors such as those which have been mentioned above, constitute in fact the vectors containing the said recombinant DNAs. This will again be the case where reference is made to xe2x80x9cdefective recombinant virusesxe2x80x9d, when the elements of the vectors associated with the recombinant DNA of the invention will be derived from a defective genome of adenovirus. These defective recombinant viruses are advantageously used for the transformation of transformable cell lines from superior eucaryotes (notably of human or animal origin) themselves including a distinct sequence of nucleotides apt to complement the part of the genome of the adenovirus which is missing from the aforesaid vector, the said distinct sequence preferably being incorporated into the genome of the cells of the said cell line.
As a prefered example of such cell lines, one might mention the line 293, a human embryonic kidney line which contains, integrated into its genome, the first eleven percent of the left end of the genome of an Ad5. This permits the complementing of the defective recombinant viruses which have deletions of this region (10).
The use of these systems: defective recombinant virus vectorxe2x80x94cells containing a sequence capable of complementing the defective recombinant viruses, is of a very particular interest, when the nucleotide sequence contained in the insertion nucleic acid of the recombinant DNA codes for a protein which, when it is expressed in the natural cellular host under the control of its natural promoter, is excreted into the culture medium of this natural cellular host.
The S gene of the genome of the virus of hepatitis B constitutes in this regard a nucleotide sequence of particular interest, and this for several reasons. On the one hand, the product of the expression of the S gene in the cells which express it, HBsAg (11, 12), is secreted into the cellular supernatant in the form of particles which are easy to detect and to quantify by radio-immunology, which permits a precise evaluation of the capacity of expression of the viral vector. On the other hand, the invention provides a recombinant viral vector permitting the study of the expression of the genes of the HBV at the level of transcription as well as that of translation, which is all the more interesting in that until now there had not existed a cell culture system capable of propagating the hepatitis B virus (HBV). And lastly, the cellular infection by the adenovirus-HBV recombinant virus illustrates particularly well the methodological basis of a process for the manufacture of a vaccine against a given pathologic agent (in this case the hepatitis B virus in the example under consideration). Another nucleotide sequence of the genome of the hepatitis B virus of particular interest is the S gene along with its pre-S2 region which codes for the HBs antigen and for a receptor of polymerized human serum albumin (nHSA) (25, 26).
It goes without saying that one may substitute for the S gene into the recombinant DNA, any other nucleotide sequence coding for a distinct antigen-protector against another given pathogenic agent, especially when this distinct antigen-protector is itself normally susceptible of being secreted by the cells transformed by the recombinant DNA. It equally goes without saying that in the recombinant DNA the S gene and the pre-S2 region may be replaced any other nucleotide sequence coding for a distinct antigen-protector against another given pathogenic agent, especially when this distinct antigen-protector is itself normally susceptible of being secreted by the cells transformed by the recombinant DNA. The nucleotide sequence coding for this distinct antigen-protector may possibly be inserted into the recombinant DNA in phase with another gene, for example the HBsAg antigen, if and when that other gene may be used as the xe2x80x9clocomotivexe2x80x9d for the promotion of the excretion equally of this distinct antigen, notably in the form of a hybrid protein. As an example of the distinct antigens susceptible of being thus produced (if need be, in the hybrid protein form), the structural glyco-proteins of the Epsteim-Barr virus may be mentioned.
The first nucleotides of the nucleotide sequence coding for a specific polypeptide (a xe2x80x9csimplexe2x80x9d or hybrid protein) are placed, notably by construction, as close as possible to this promoter, notably to the xe2x80x9cTATA boxxe2x80x9d, which is characteristic of the promoter, it being understood however that the nucleotide sequence between the promoter and the ATG initiator of the nucleotide sequence coding for the said specific polypeptide should in general contain the triplets coding for the non-translated 5xe2x80x2 end of the messenger RNA corresponding normally to the coding sequence and containing the matching sequences to the ribosomes necessary to an effective translation. This untranslated 5xe2x80x2 end of the messenger RNA could also be replaced by the untranslated 5xe2x80x2 end of a messenger RNA distinct from that normally associated with a specific coding sequence. For example, one may, in the case of the S gene, replace the untranslated 5xe2x80x2 end containing the pre-S gene or juxtaposing this with the untranslated 5xe2x80x2 end of the messenger RNA of the T antigen of SV40. But it has also been noted that when using a DNA sequence containing the S and pre-S2 regions of the genome of the hepatitis B virus under the control of the strong promoter, E1A, it is possible to obtain the expressions both of the pre-S2 and the S regions. Any other untranslated 5xe2x80x2 end of messenger RNA may be used if it is compatible with the other similar chosen end.
It is advantageous that the distance between the TATA box of the promoter and the initiation site of the messenger RNA should be around 30 nucleotides.
The E1A promoter of the recombinant DNA according to the invention and more generally the vector according to the invention using the more important parts of the adenovirus genome are preferably derived from an adenovirus belonging to category C, as defined by TOOZE. These adenoviruses have the known property of not being oncogenic. The sub-types Ad2 or Ad5 of this category of adenovirus are characterized by an important transforming power. The use of this latter type of recombinant DNA is therefore particularly recommended, when the desired expression product is intended for the production of antigen-protectors, notably the active principles of vaccines. This will be even more true in the case where whole adenoviruses, and even infectious, will be used as the active principles of live vaccines, notably under the conditions which will be spelled out further on.
The invention naturally equally concerns the cell lines, notably of human or animal origin, which are transformed by the recombinant DNAs as defined hereabove and which are rendered capable of synthesizing a polypeptide coded by the nucleotide sequence (or the said nucleotide sequences) contained in these recombinant DNAs and placed under the direct control of said promoter.
The invention concerns more particularly yet the cell lines transformed with the recombinant DNA conforming to the invention and in addition characterized in that the cells of the cell lines themselves contain a distinct sequence of nucleotides capable of complementing the part of the adenovirus genome which is missing from the aforesaid vector, the said distinct sequence being preferably incorporated into the genome of the cells of the said cell line.
In this regard, the line 293 already mentioned above, after having been transformed by the recombinant DNAs, constitutes a prefered cellular culture according to the invention. Due to the complementation sequence contained by the cells of this line, a major viral multiplication within these cells is observed and, by way of consequence, an equally multiplied expression of the sequence coding for the predetermined polypeptide. In the first case where this coding sequence is the S gene, a high production is obtained of the HBsAg antigens excreted into the culture medium of these cells. In the second case where the coding sequences are constituted of the S gene and the pre-S2 region of the hepatitis B virus, the recombinant adenovirus directs in vitro the synthesis of the HBsAg particles possessing an activity for the pHSA receptor. Injected into rabbits, this recombinant virus produces anti-HBsAg and anti-pHSA antibodies. This shows the possibility of using the recombinant adenovirus to express a gene both in vitro and in vivo. The same vectors may be used for the transformation of Vero cells under analogue conditions.
The vectors containing the recombinant DNA according to the invention may equally be used for the transformation of cells not possessing themselves the complementation sequence under the conditions which have been indicated hereabove. It may then be necessary to proceed to a co-transformation of these latter types of cells, on the one hand, with a vector containing the recombinant DNA according to the invention, and on the other, with a non-defective adenovirus or a distinct recombinant DNA containing the adenovirus sequences which are missing from the recombinant vector conforming to the invention. It may certainly be observed in this latter case a simultaneous production of HBsAg antigens (when the coding sequence contains the S gene) and of the adenovirus replicated and liberated by the cells thus transformed. The antigen-protector formed may however be separated from the viral suspension, if need be, for example by bringing the culture medium into contact with anti-adenovirus antibodies, preferably immobilized on a solid support, such as reticulated agarose, sold under the designation, SEPHAROSE. In any case, the presence of residual quantities of the virus in the vaccinating preparation is only of a relatively minor importance. Indeed, the adenovirus has only a weak pathogenic power in humans, causing no more than benign respiratory infections.
The small importance of the pathogenic power of the adenoviruses, more particularly of those belonging to group C of the human adenoviruses, allows the contemplation of xe2x80x9cliving vaccinesxe2x80x9d. These could be constituted of infectious adenoviruses modified at the level of the E3 region by the insertion of the recombinant DNA according to the invention in the non-essential part of the adenovirus. In this regard, the fact must be emphasized that the human adenoviruses of the C group have never proved to be tumorigenic in animals (3). These vectors or viruses will be of a very particular interest for the transformation of Vero cells, the non-tumorigenic character of which has been firmly established. For this reason they constitute a line of animal origin particularly favorable to the production of products for human use.